Antimicrobial Peptide Recognition of Bacterial Membranes

334a

Monday, March 7, 2011

1813-Pos Board B723 Antimicrobial Peptides Which Exhibit Lipid Composition Dependent Membrane Binding and Sequence Dependent Efficacy of Bacteriolysis Luba Arotsky, Emmanuel Yawson, Laura Hagens, Michael Urban, Gregory A. Caputo. Antimicrobial peptides are a class of short, amphiphilic peptides which are part of the innate immune system of most higher organisms. These molecules act as a first line of defense against bacterial infection, exhibiting selective recognition of bacterial pathogens over host cells and rapid killing kinetics. The studies presented focus on the antimicrobial peptide C18G which is derived from the cterminal sequence of a human platelet factor protein. This peptide has shown high efficacy in the low micromolar range against both gram-positive and gram-negative species which was maintained upon an amino acid substitution to facilitate monitoring via fluorescence spectroscopy (Y3W). The initial experiments focused on the binding affinities of sequence-substituted C18G variants in which the cationic Lysine residues were replaced with either Arginine or Histidine. All variants of the peptide exhibited high affinity for lipid membranes that contain anionic lipids (PG or PS) and a significant decrease in affinity for bilayers composed only of zwitterionic lipids (PC or PC/PE mixtures). All peptides exhibited similar binding affinities when the solution pH was lowered to pH4 or raised to pH10, indicative of a multi-component binding. Additionally, all peptides were shown to adopt a predominantly alpha-helical conformation when bound to lipid vesicles or detergent micelles. A dramatically different profile was observed when these peptides were were tested for bacterial membrane permeabilization. The Lys and Arg variants exhibited an expected dose-dependent permeabilization of both the outer and inner membranes of E.coli while the His variant showed no increase in membrane permeability. This pattern held true for the disruption of S.aureus membrane potential by the same set of peptides. These results are indicative of decoupling the binding and killing events which argues against a nonspecific, membrane aggregate mechanism of action. 1814-Pos Board B724 The Effect of Point Mutations on the Secondary Structure and Membrane Interaction of Antimicrobial Peptide Anoplin Amy Won, Stahs Pripotnev, Annamaria Ruscito, Anatoli Ianoul. Isolated from the venom sac of solitary spider wasp, Anoplius samariensis, Anoplin (GLLKRIKTLL-NH2) is the smallest, amphiphathic, linear a-helical antimicrobial peptide found naturally to date. It has a broad-spectrum activity against both Gram-positive and Gram-negative bacteria and exhibits low hemolytic activity towards human erythrocytes. Previous studies suggested antimicrobial and hemolytic properties of Anoplin are strongly correlated with its physicochemical properties. Two derivatives, Anoplin-8K (Ano8K, GLLKRIKKLL-NH2) and Anoplin-1K5V8K (Ano1K5V8K, KLLKVIKKLLNH2), were found previously that possess increased antimicrobial activity while retaining low hemolytic activity. In the present work, native Anoplin, Ano8K and Ano1K5V8K were further investigated using UV resonance Raman (UVRR) spectroscopy, Langmuir monolayer technique and carboxyfluorescein (CF) leakage assay. UVRR data indicated that all three peptides adopt predominantly a random coil conformation in aqueous buffer solution. The alpha-helical content of all three peptides increases in membrane mimicking trifluoroethanol with Ano1K5V8K having the highest alpha-helix percentage, followed by Ano8K and Anoplin. Critical micelle concentrations were found to decrease in a sequence Ano1K5V8K, Anoplin and Ano8K. Critical pressure of insertion was found to be greater for anionic DPPG monolayer than for zwitterionic DPPC monolayer indicating preferential adsorption of all three peptides to DPPG. Finally, CF leakage data indicated membrane lytic activities of Anoplin toward bacterial model membrane (E. coli, B. subtilis and S. aurues) correlate with peptide helicity and amphipathicity. 1815-Pos Board B725 Antimicrobial Peptide Recognition of Bacterial Membranes Marc-Antoine Sani, David I. Fernandez, John D. Gehman, Frances Separovic. In the face of increasing clinical resistance to existing antibiotic drugs, antimicrobial peptides (AMP) are seen as a source of new antibiotics. Since these AMP have high affinities for bacterial membranes, but with highly variable activities over different strains, a multidisciplinary approach combining solidstate NMR, microbiology and lipidomics has been taken to decipher their specific recognition. Most studies of AMP have been carried out using model membrane systems, such as phosphatidylcholine (PC) and phosphatidylglycerol (PG) bilayers, which bear questionable resemblance to bacterial membranes. We have expanded these studies to include other bacterial phospholipids, e.g. phosphatidylethanolamine, cardiolipin (CL) and natural

lipid extracts. Model membranes prepared from bacterial lipid extracts and live pathogenic bacteria are being used to help determine the mechanism of action of a class of AMP obtained from Australian tree frogs. The aims are to determine at which growth stage of the bacteria the peptides are active and to correlate with the membrane composition at each stage using mass spectrometry of the extracted phospholipids (lipidomics). Ongoing solid-state NMR experiments are providing in vivo AMP activity spectra at atomic resolution. This includes monitoring of phospholipid-peptide association and identification of specific bacterial features, such as acyl chain lipid composition, as critical mechanisms of antibiotic resistance. We report AMPs that have shown a better affinity for CL-containing vesicles, than for PC or a PC/PG mixture, and promote lamellar lipid organization to break into isotropic, as yet unsolved, structures. These correlate with the lower minimum inhibitory concentration assays done on a range of bacteria and circular dichroism experiments of AMP with specific lipid composition as those used in preliminary in vivo NMR experiments. 1816-Pos Board B726 Neutron Reflectivity Studies of the Antimicrobial Peptide Maculatin 1.1 in Supported Lipid Bilayers David I. Fernandez, Anton P. Le Brun, Paramjit Bansal, Michael James, Francis Separovic. Maculatin 1.1 is a member of a small group of a-helical antimicrobial peptides isolated from the skin secretions of Australian amphibians. Previous studies using solid-state NMR, surface plasmon resonance, quartz crystal microbalance, calorimetry and fluorescence measurements have revealed that this peptide acts via different mechanisms dependent on concentration and membrane charge, which lead to the disruption of bacterial membranes. Neutron reflection is a method that provides information about the distribution of materials on the axis perpendicular to a membrane surface (the z-axis). Neutron scattering can discriminate between hydrogen and its isotope deuterium, making neutron reflection a powerful tool for dissecting how lipid, protein and solvent relate to one another along the z-axis and provides a method in which certain components can be highlighted or made invisible by choosing the correct isotopic contrast. In this case deuterated lipid chains and a hydrogenated peptide were used. The interaction of maculatin 1.1 in solid-supported lipid bilayers that mimic the essential charge characteristics of eukaryotic (neutral) and prokaryotic (anionic) membranes were studied using neutron reflection. The peptide was found to bind to both bilayers but the action of the peptide was different between each bilayer. When maculatin 1.1 bound to neutral bilayers the bilayer structure was lost and lipid order diminished whereas when maculatin 1.1 bound to negatively charged bilayers lipid order increased and the bilayer structure was retained suggesting that maculatin 1.1 formed pores when bound to negatively charged bilayers. 1817-Pos Board B727 Studies of Membrane-Active Peptides Using Neutron Diffraction Juan C. Cruz, Mihaela Mihailescu, William Wimley, Kalina Hristova. Biomembrane-active peptides and particularly those capable of translocating have attracted considerable attention in the biotech community as they are likely to enable a number of emerging platforms for drug and gene delivery. A major limitation in the design of such active peptides is the limited knowledge of their mode of action. We are working to address this knowledge gap by directly observing these peptides while interacting with lipid bilayers. The goal of the study is to determine the disposition of the peptides using neutron diffraction, and identify key differences in the disposition of translocating and inactive peptides. This is accomplished through specific deuteration of selected amino acids in the two peptides. The experiments also reveal the effect of the peptides on bilayer structure. 1818-Pos Board B728 Molecular Basis for Anticancer Activity of Host Defense Peptides Andrey Ivankin, Paul A. Adamczyk, Binill H. Shah, David Gidalevitz. The high toxicity of most anticancer chemotherapeutic drugs and their deactivation by multidrug resistant phenotypes motivated an extensive search for drugs with new modes of action. Host defense peptides (HDPs) represent a promising class of natural-source drugs with a distinct mode of action, decreased likelihood of resistance development, and low intrinsic cytotoxicity. However, the development of antitumoral HDP-based compounds has been hindered by poor understanding of the molecular mechanisms of the HDPs’ selective killing of cancer cells. Accumulating evidence indicates that the outer leaflet of tumor cell plasma membranes is enriched in anionic lipids such as phosphatidylserine (PS) and various gangliosides that may render the cancer cells susceptible to cationic HDPs. Here we report results of a combined AFM-X-ray study aimed at the understanding an impact of elevated levels of